PROJECT SUMMARY Acute kidney injury (AKI) is a major unmet medical need due to the lack of effective pharmacological treatment options and significant healthcare burden of the disease. The fact that AKI mortality remains at 50-80% and has not improved in decades underscores the critical need for better treatments. Ischemia-reperfusion injury (IRI) and nephrotoxic agents (e.g., cisplatin) are critical causative factors in AKI pathophysiology. IRI is a major challenge during organ transplantation and cardiothoracic, vascular and general surgery. A wide range of pathological processes including oxidative stress, inflammation and activation of cell death programs, such as apoptosis and necrosis contribute to tissue injury and renal dysfunction in AKI. We have selected p53 and CXCR4 as potential targets for combination therapies that affect important metabolic and proinflammatory pathways in AKI. This proposal addresses the urgent need for new renoprotective treatments by developing a novel integrated siRNA delivery platform capable of selective combined inhibition of p53 and CXCR4 in AKI. The objective is to develop innovative polymer-siRNA conjugates for efficient and safe delivery of CXCR4 antagonist and anti-p53 siRNA (sip53) to proximal tubule cells of the injured kidneys. To achieve the objective, we will use polymeric CXCR4 antagonist (PCX) as the sip53 carrier and test if delivery of PCX-sip53 conjugates to CXCR4 overexpressing proximal tubule reverses ATP depletion and ameliorates inflammation, renal injury and dysfunction. We will accomplish the overall objective by pursuing the following specific aims. In aim 1, we will synthesize covalent PCX-siRNA conjugates using different linker chemistries and different molecular weight and chemical composition of PCX. We will establish the conjugate safety, CXCR4 antagonism, and the ability to deliver siRNA to proximal tubule cells due to the beneficial effect of PCX on facilitating cytoplasmic delivery of the siRNA. In aim 2, we will validate the proximal tubule-selective delivery of the conjugates by conducting comprehensive pharmacokinetic and biodistribution study and evaluating therapeutic efficacy in IRI and cisplatin models of AKI in mice. In aim 3, we will assess in detail the therapeutic efficacy of the best-performing PCX- sip53 conjugate. Using a set of mechanistic studies in wild type mice and in mice with proximal tubule-selective p53 knockout, we will illuminate how the combined inhibition of p53 and CXCR4 ameliorates the pathophysiology of AKI. Overall, the innovative design of the PCX-siRNA conjugates will establish a widely applicable approach for specific delivery of therapeutic siRNA to the injured kidney. The PCX-siRNA conjugate design will also have broader impact and serve as a prelude to efficacious treatment approaches in various other ischemic diseases including myocardial ischemia and stroke.